M-MLV Reverse Transcriptase(Glycerol free) HYA213
This product is derived from Moloney Murine Leukemia Virus, which is an RNA-dependent DNA polymerase that lacks 3'→5' exonuclease activity and has its RNase H activity removed by mutation. This enzyme is a new reverse transcriptase obtained by multiple point mutations based on M-MLV (RNase H-). It can withstand a reaction temperature of 50°C and can be used for reverse transcription of RNA templates with secondary structures to enhance affinity with the template. This product can be used for first-strand cDNA synthesis, second-strand cDNA synthesis, RNA primer extension, RT-PCR, RT-qPCR and cDNA library construction.
Components
|
Component |
10 KU |
100 KU |
2000 KU |
|
200 U/μL M-MLV Reverse Transcriptase(Glycerol free) |
0.05 mL |
0.5 mL |
10 mL |
|
5×Reverse Reverse Transcriptase Buffer |
0.5 mL |
5 mL |
10×10 mL |
Storage
Store at -25°C~ -15°C.
Unit Definition
1 activity unit (U) is defined as the amount of enzyme required to convert 1 nmol of dTTP into acid-insoluble substances at 37°C for 10 min using Poly (rA)·Oligo (dT) as template/primer.
Quality Control
1. Protein purity(SDS-PAGE): ≥95%;
2. Endonuclease residue: 200 U of this product and 1 μg λDNA reacted at 37℃ for 16 hours, and the electrophoretic band of DNA did not change;
3. Exonuclease residue: 200 U of this product and 1 μg λ-HindIII digest DNA reacted at 37℃ for 16 hours, and the electrophoretic band of DNA did not change;
4. RNase residue: 200 U of this product and 1.6 μg MS2RNA reacted at 37℃ for 4 hours, and the electrophoretic band of RNA did not change;
5. E.coli genome residue: The residual nucleic acid in 600 U of this product was detected by TaqMan qPCR specific for E.coli 16s rDNA, and the E.coli genome residue was less than 10 copies;
Instructions
1. Reaction system
|
Components |
Volume |
|
Template RNA* |
optional |
|
Oligo(dT) 18 (50uM) or Random Primer mix(50ng/μL) |
1 μL |
|
dNTP Mix (10mM each) |
1 μL |
|
RNase Inhibitor (40U/uL) |
1 μL |
|
200 U/μL M-MLV Reverse Transcriptase(Glycerol free) |
1 μL |
|
5×Reverse Reverse Transcriptase Buffer |
4 μL |
|
Nuclease-free Water |
Up to 20 μL |
*The amount of template RNA added can be referred to the following table
|
Total RNA |
10 pg~5μg |
|
Poly(A) RNA |
10 pg~500ng |
2. Reaction Program
|
Temperature |
Time |
|
25 °C* |
5 mins |
|
50 °C** |
45 min*** |
|
85 °C |
2 min |
*If using Random Primer Mix, add this incubation step; omit this step when using Oligo(dT)18 or Gene Specific Primer.
**If the template has a complex secondary structure or a high GC region, the reaction temperature can be increased to 55℃ to help increase the yield.
***Prolonging the reverse transcription time can help obtain longer cDNA (>5 kb)
Notes
1. To prevent RNase contamination, please keep the experimental area clean and wear clean gloves and masks when operating. The consumables such as centrifuge tubes and pipette tips used in the experiment must be RNase-free.
2. cDNA products can be stored at -20℃ or -80℃ or used immediately for PCR (qPCR) reactions.
3. The volume of cDNA products used for PCR (qPCR) reactions is recommended not to exceed 1/10 of the PCR reaction volume.